Non-invasive diagnostic imaging techniques for examining the microscopic structure of tissue is desirable for the skin, where the standard examination practice of biopsy can lead to scarring. Two techniques that have garnered much interest in recent years for dermatology use are reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM). The optical sectioning capability of RCM allows in vivo, high resolution morphological images of skin. MPM also has inherent optical sectioning capabilities and allows sensitive in vivo imaging at great depths.
Multiphoton signals include signals from multiphoton fluorescence and sum frequency generation. Multiphoton fluorescence occurs when two or more photons of relatively lower energy are simultaneously absorbed by and excite a fluorophore, causing emission of a fluorescence photon at a higher energy than the excitation photons. Sum frequency generation occurs when two or more photons interact with a nonlinear material and combine to form a new photon with a multiple of the frequency and a fraction of the wavelength of the initial photons. Both multiphoton fluorescence and sum frequency generation is localized to where the light source, for example a femtosecond laser, is focused and provides a high flux of photons.
Different MPM excitation mechanisms are sensitive to different biochemical compositions of the tissue. For example, two-photon fluorescence (TPF) signals arise from endogenous fluorophores of skin components such as elastin, NAD(P)H, and keratin; while second harmonic generation (SHG) is sensitive to non-centrosymmetric structures such as collagen.
As there is less scattering and absorption of the near infrared light used in MPM, there is deeper penetration as well as less photo-damage to the tissue. Combining both RCM and MPM imaging (RCM/MPM imaging) potentially allows greater clinical diagnostic utility as complementary information can be revealed using the two techniques. RCM/MPM imaging has been applied in ex vivo and in vivo studies. For clinical application, in vivo imaging is preferred over ex vivo imaging because it does not necessitate tissue removal. It also leaves the tissue in its native state, whereas ex vivo tissue can be subject to biochemical/structural changes due to the degradation of the sample, tissue contraction, and elimination of living tissue dynamics such as blood perfusion and oxygenation.
In vivo skin imaging is complicated because patient motion must be mitigated, and often multiple or large lesions must be examined. Some in vivo MPM systems have imaging rates varying from 1 s to 24 s per frame for titanium sapphire laser systems based at 800 nm, and 0.5 s to 2 s per frame for chromium-forsterite laser systems based around 1250 nm. These slow imaging rates can result in blurred images and prolonged imaging times. Fast imaging rate is important for decreasing blurring effects and reducing patient imaging times.
Conventional MPM systems with multiple imaging modes typically employ a dedicated photomultiplier tube (PMT) for each imaging modality. For example, MPM systems that detect both TPF and SHG signals use a dichroic mirror to separate light emitted from the sample into the two signals and direct the signals to respective PMTs. Filters are also typically located in the emitted light path before each PMT. Optical components such as mirrors and filters decrease signal strength by absorbing and/or reflecting some of the emitted light and also by causing the emitted light path to be longer in order to accommodate placement of the optical components. A further disadvantage is that changing wavelengths to switch between multiphoton imaging modes requires changing out or adjusting these optical components, increasing the complexity of the system as well as increasing the time and labour associated with imaging in multiple modes.
Selective photothermolysis is based on the selective absorption of pulsed light radiation by the targeted chromophores. In selective photothermolysis based skin phototherapy, for example, the therapeutic laser simultaneously illuminates a large volume of tissue. For successful therapy, heat generation must be restricted to the targeted chromophores only, which is done by selecting a laser wavelength at which the targeted chromophores have much higher light absorption than non-targeted components. This type of skin phototherapy has been somewhat successful in treating pigmented skin diseases and in permanent hair removal. However, side effects and inefficiencies occur when there is less selectivity of light absorption by the target chromophores.
Apparatus and methods of multiphoton microscopy that address one or more disadvantages of conventional systems are desirable.